Post-transcriptional regulation of soluble guanylyl cyclase expression in rat aorta

We investigated the molecular mechanism of cyclic GMP-induced down-regulation of soluble guanylyl cyclase expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an allosteric activator of this enzyme, decreased the expression of soluble guanylyl cyclase alpha(1) subunit mRNA and protein. This effect was blocked by the enzyme inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase alpha(1) mRNA-degrading activity was increased in protein extracts from YC-1-exposed aorta and was attenuated by pretreatment with actinomycin D and NS2028. Gelshift and supershift analyses using an adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region of the alpha(1) mRNA and a monoclonal antibody directed against the mRNA-stabilizing protein HuR revealed HuR mRNA binding activity in aortic extracts, which was absent in extracts from YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this decrease was prevented by NS2028. Similarly, down-regulation of HuR by RNA interference in cultured rat aortic smooth muscle cells decreased alpha(1) mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha(1) mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha(1) mRNA and lowering alpha(1) subunit expression as a negative feedback response.     

DMT1 expression is increased in the lungs of hypotransferrinemic mice

Despite a lack of transferrin, hypotransferrinemic (Hp) mice demonstrate an accumulation of iron in peripheral organs including the lungs. One potential candidate for such transferrin-independent uptake of iron is divalent metal transporter-1 (DMT1), an established iron transporter. We tested the hypothesis that increased concentrations of iron in the lungs of Hp mice are associated with elevations in DMT1 expression. With the use of inductively coupled plasma emission spectroscopy, measurements of nonheme iron confirmed significantly elevated concentrations in the lung tissue of Hp mice relative to the wild-type mice. Western blot analyses for the expression of two isoforms of DMT1 in the Hp mice relative to the wild-type animals demonstrated an elevation for the isoform that lacks an iron-responsive element (IRE) with significant decrements in the expression of +IRE DMT1. With the use of immunohistochemistry, -IRE DMT1 was localized to both airway epithelial cells and alveolar macrophages in wild-type mice. Staining appeared increased in both types of cells in the Hp mice. Elevated concentrations of both tissue nonheme iron and expression of -IRE DMT1 in the Hp mice were associated with increased quantities of -IRE mRNA. There was no difference between wild-type and homozygotic Hp mice in the amount of mRNA for DMT1 +IRE. We conclude that differences between Hp and wild-type mice in nonheme iron concentrations were accompanied by increases in the expression of -IRE DMT1. Increased expression of -IRE DMT1 in the lungs of the Hp mice could be responsible for elevated concentrations of the metal in these tissues.     

肥胖对小鼠十二指肠 DMT1和 FPN1表达的影响

目的：观察肥胖对小鼠十二指肠二价金属离子转运体（divalent metal transporter 1,DMT1）mRNA、膜铁转运蛋白（ferroportin1,FPN1）mRNA及蛋白表达的变化,探讨肥胖影响铁吸收的机制。方法 C57BL／6J小鼠随机分为正常对照组和肥胖模型组,每组6只,通过喂养高脂饲料喂养建立肥胖模型,对照组采用普通饲料饲养,实验干预期14周。建模完成后,采用实时荧光定量PCR方法检测小鼠十二指肠DMT1、FPN1 mRNA 的表达,用Western blot检测小鼠十二指肠FPN1蛋白表达。结果与对照组小鼠相比,肥胖模型组小鼠十二指肠DMT1、FPN1 mRNA表达以及FPN1蛋白表达水平降低,差异具有统计学意义（ P ＜0.05）。结论肥胖会下调机体十二指肠DMT1、FPN1的表达,导致铁吸收不良,为进一步研究肥胖引起铁缺乏机制提供理论和实验依据。     

DMT1 and FPN1 expression during infancy: developmental regulation of iron absorption

Two iron transporters, divalent metal transporter1 (DMT1) and ferroportin1 (FPN1) have been identified; however, their role during infancy is unknown. We investigated DMT1, FPN1, ferritin, and transferrin receptor expression, iron absorption and tissue iron in iron-deficient rat pups, iron-deficient rat pups given iron supplements, and controls during early (day 10) and late infancy (day 20). With iron deficiency, DMT1 was unchanged and FPN1 was decreased (-80%) at day 10. Body iron uptake, mucosal iron retention, and total iron absorption were unchanged. At day 20, DMT1 increased fourfold and FPN1 increased eightfold in the low-Fe group compared with controls. Body iron uptake and total iron absorption were increased, and mucosal iron retention was decreased with iron deficiency. Iron supplementation normalized expression levels of the transporters, body iron uptake, mucosal iron retention, and total iron absorption of the low-Fe group to those of controls at day 20. In summary, the molecular mechanisms regulating iron absorption during early infancy differ from late infancy when they are similar to adult animals, indicating developmental regulation of iron absorption.     

Post-transcriptional regulation of human cathepsin L expression

The expression of cathepsin L, a lysosomal protease, is known to be elevated in cancer and other pathologies. Multiple splice variants of human cathepsin L with variable 5'UTRs exist, which encode for the same protein. Previously we have observed that variant hCATL A (bearing the longest 5'UTR) was translated in vitro with significantly lower efficiency than variant hCATL AIII (bearing the shortest 5'UTR). Contrary to these findings, results of the present study reveal that in cancer cells, hCATL A mRNA exhibits higher translatability in spite of having lower stability than AIII. This is the first report demonstrating a highly contrasting trend in translation efficiencies of hCATL variants in rabbit reticulocytes and live cells. Expression from chimeric mRNAs containing 5'UTRs of A or AIII upstream to luciferase reporter cDNA established the A UTR to be the sole determinant for this effect. Transient transfections of bicistronic plasmids and mRNAs confirmed the presence of a functional Internal Ribosome Entry Site in this UTR. Our data suggest that differential stability and translation initiation modes mediated by the 5'UTRs of human cathepsin L variants are involved in regulating its expression.